RESUMO
Necroptosis, necrosis characterized by RIPK3-MLKL activation, has been proposed as a mechanism of doxorubicin (DOX)-induced cardiomyopathy. We showed that rapamycin, an mTORC1 inhibitor, attenuates cardiomyocyte necroptosis. Here we examined role of MLKL in DOX-induced myocardial damage and protective effects of rapamycin. Cardiomyopathy was induced in mice by intraperitoneal injections of DOX (10 mg/kg, every other day) and followed for 7 days. DOX-treated mice showed a significant decline in LVEF assessed by cardiac MRI (45.5 ± 5.1% vs. 65.4 ± 4.2%), reduction in overall survival rates, and increases in myocardial RIPK3 and MLKL expression compared with those in vehicle-treated mice, and those changes were prevented by administration of rapamycin (0.25 mg/kg) before DOX injection. In immunohistochemical analyses, p-MLKL signals were detected in the cardiomyocytes of DOX-treated mice, and the signals were reduced by rapamycin. Mlkl+/- and Mlkl-/- mice were similarly resistant to DOX-induced cardiac dysfunction, indicating that a modest reduction in MLKL level is sufficient to prevent the development of DOX-induced cardiomyopathy. However, evidence of cardiomyocyte necrosis assessed by C9 immunostaining, presence of replacement fibrosis, and electron microscopic analyses was negligible in the myocardium of DOX-treated mice. Thus, MLKL-mediated signaling contributes to DOX-induced cardiac dysfunction primarily by a necrosis-independent mechanism, which is inhibitable by rapamycin.
Assuntos
Cardiomiopatias , Doxorrubicina , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Necroptose , Proteínas Quinases , Proteína Serina-Treonina Quinases de Interação com Receptores , Sirolimo , Animais , Doxorrubicina/efeitos adversos , Proteínas Quinases/metabolismo , Sirolimo/farmacologia , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/prevenção & controle , Cardiomiopatias/patologia , Cardiomiopatias/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Necroptose/efeitos dos fármacos , Masculino , Camundongos , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/toxicidadeRESUMO
Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.
RESUMO
Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.
Assuntos
Lectinas , Polissacarídeos , Proteínas Recombinantes , Proteínas Recombinantes/química , Polissacarídeos/química , Polissacarídeos/análise , Lectinas/química , Glicosilação , Automação Laboratorial/métodosRESUMO
Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galß1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.
RESUMO
Necroptosis, a form of necrosis, and alterations in mitochondrial dynamics, a coordinated process of mitochondrial fission and fusion, have been implicated in the pathogenesis of cardiovascular diseases. This study aimed to determine the role of mitochondrial morphology in canonical necroptosis induced by a combination of TNFα and zVAD (TNF/zVAD) in H9c2 cells, rat cardiomyoblasts. Time-course analyses of mitochondrial morphology showed that mitochondria were initially shortened after the addition of TNF/zVAD and then their length was restored, and the proportion of cells with elongated mitochondria at 12 h was larger in TNF/zVAD-treated cells than in non-treated cells (16.3 ± 0.9% vs. 8.0 ± 1.2%). The knockdown of dynamin-related protein 1 (Drp1) and fission 1, fission promoters, and treatment with Mdivi-1, a Drp-1 inhibitor, had no effect on TNF/zVAD-induced necroptosis. In contrast, TNF/zVAD-induced necroptosis was attenuated by the knockdown of mitofusin 1/2 (Mfn1/2) and optic atrophy-1 (Opa1), proteins that are indispensable for mitochondrial fusion, and the attenuation of necroptosis was not canceled by treatment with Mdivi-1. The expression of TGFß-activated kinase (TAK1), a negative regulator of RIP1 activity, was upregulated and the TNF/zVAD-induced RIP1-Ser166 phosphorylation, an index of RIP1 activity, was mitigated by the knockdown of Mfn1/2 or Opa1. Pharmacological TAK1 inhibition attenuated the protection afforded by Mfn1/2 and Opa1 knockdown. In conclusion, the inhibition of mitochondrial fusion increases TAK1 expression, leading to the attenuation of canonical necroptosis through the suppression of RIP1 activity.
Assuntos
Dinâmica Mitocondrial , Necroptose , Ratos , Animais , Regulação para Baixo , Necrose/metabolismo , Mitocôndrias/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Lectinas , Polissacarídeos/química , Espectrometria de MassasRESUMO
Diabetes mellitus is one of the major causes of ischemic and nonischemic heart failure. While hypertension and coronary artery disease are frequent comorbidities in patients with diabetes, cardiac contractile dysfunction and remodeling occur in diabetic patients even without comorbidities, which is referred to as diabetic cardiomyopathy. Investigations in recent decades have demonstrated that the production of reactive oxygen species (ROS), impaired handling of intracellular Ca2+, and alterations in energy metabolism are involved in the development of diabetic cardiomyopathy. AMP deaminase (AMPD) directly regulates adenine nucleotide metabolism and energy transfer by adenylate kinase and indirectly modulates xanthine oxidoreductase-mediated pathways and AMP-activated protein kinase-mediated signaling. Upregulation of AMPD in diabetic hearts was first reported more than 30 years ago, and subsequent studies showed similar upregulation in the liver and skeletal muscle. Evidence for the roles of AMPD in diabetes-induced fatty liver, sarcopenia, and heart failure has been accumulating. A series of our recent studies showed that AMPD localizes in the mitochondria-associated endoplasmic reticulum membrane as well as the sarcoplasmic reticulum and cytosol and participates in the regulation of mitochondrial Ca2+ and suggested that upregulated AMPD contributes to contractile dysfunction in diabetic cardiomyopathy via increased generation of ROS, adenine nucleotide depletion, and impaired mitochondrial respiration. The detrimental effects of AMPD were manifested at times of increased cardiac workload by pressure loading. In this review, we briefly summarize the expression and functions of AMPD in the heart and discuss the roles of AMPD in diabetic cardiomyopathy, mainly focusing on contractile dysfunction caused by this disorder.
RESUMO
The association between altered glycosylation of MUC1 and various disease events has sparked significant interest. However, analytical technologies to investigate the disease-related glycoforms of endogenous MUC1 in blood and tissue specimens are limited. Therefore, we devised a reliable technique for differential analysis of endogenous MUC1 glycoforms based on an antibody-assisted lectin microarray. Its highly sensitive detection aids in analyzing soluble MUC1 from relatively small amounts of serum via a simple enrichment process. Micro-/macro-dissection of the MUC1-positive region is combined with glycoform analysis of the membrane-tethered MUC1. Thus, we have optimized the protocol for sample qualification using immunohistochemistry, sample pretreatment for tissue sections, protein extraction, purification via immunoprecipitation, and the antibody-overlay lectin microarray, which are sequentially essential for differential glycoform analysis of endogenous MUC1.
Assuntos
Lectinas , Mucina-1 , Lectinas/metabolismo , Mucina-1/metabolismo , Anticorpos , Análise em Microsséries/métodos , Imuno-HistoquímicaRESUMO
Aberrant glycosylation is an important factor in facilitating tumor progression and therapeutic resistance. In this study, using Wisteria floribunda agglutinin (WFA), we examined the expression of WFA-binding glycans (WFAG) in cholangiocarcinoma (CCA). The results showed that WFAG was highly detected in precancerous and cancerous lesions of human CCA tissues, although it was rarely detected in normal bile ducts. The positive signal of WFAG in the cancerous lesion accounted for 96.2% (50/52) of the cases. Overexpression of WFAG was significantly associated with lymph node and distant metastasis (P < 0.05). The study using the CCA hamster model showed that WFAG is elevated in preneoplastic and neoplastic bile ducts as early as 1 month after being infected with liver fluke and exposed to N-nitrosodimethylamine. Functional analysis was performed to reveal the role of WFAG in CCA. The CCA cell lines KKU-213A and KKU-213B were treated with WFA, followed by migration assay. Our data suggested that WFAG facilitates the migration of CCA cells via the activation of the Akt and ERK signaling pathways. In conclusion, we have demonstrated the association of WFAG with carcinogenesis and metastasis of CCA, suggesting its potential as a target for the treatment of the disease.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Lectinas de Plantas , Polissacarídeos , Receptores de N-Acetilglucosamina , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Humanos , Lectinas de Plantas/metabolismo , Polissacarídeos/metabolismo , Polissacarídeos/química , Receptores de N-Acetilglucosamina/metabolismo , Cricetinae , Masculino , Carcinogênese/metabolismo , Carcinogênese/patologia , Metástase Neoplásica , Feminino , Pessoa de Meia-Idade , Movimento Celular/efeitos dos fármacosRESUMO
Glioma stem cell/glioma-initiating cell (GIC) and their niches are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanisms of GIC maintenance/differentiation, we performed a unique integrated proteogenomics utilizing GIC clones established from patient tumors having the potential to develop glioblastoma. After the integration and extraction of the transcriptomics/proteomics data, we found that chondroitin sulfate proteoglycan 4 (CSPG4) and its glycobiosynthetic enzymes were significantly upregulated in GICs. Glyco-quantitative PCR array revealed that chondroitin sulfate (CS) biosynthetic enzymes, such as xylosyltransferase 1 (XYLT1) and carbohydrate sulfotransferase 11, were significantly downregulated during serum-induced GIC differentiation. Simultaneously, the CS modification on CSPG4 was characteristically decreased during the differentiation and also downregulated by XYLT1 knockdown. Notably, the CS degradation on CSPG4 by ChondroitinaseABC treatment dramatically induced GIC differentiation, which was significantly inhibited by the addition of CS. GIC growth and differentiation ability were significantly suppressed by CSPG4 knockdown, suggesting that CS-CSPG4 is an important factor in GIC maintenance/differentiation. To understand the molecular function of CS-CSPG4, we analyzed its associating proteins in GICs and found that CSPG4, but not CS-CSPG4, interacts with integrin αV during GIC differentiation. This event sequentially upregulates integrin-extracellular signal-regulated kinase signaling, which can be inhibited by cyclic-RGD (Arg-Gly-Asp) integrin αV inhibitor. These results indicate that CS-CSPG4 regulates the GIC microenvironment for GIC maintenance/differentiation via the CS moiety, which controls integrin signaling. This study demonstrates a novel function of CS on CSPG4 as a niche factor, so-called "glyco-niche" for GICs, and suggests that CS-CSPG4 could be a potential target for malignant glioma.
Assuntos
Proteoglicanas de Sulfatos de Condroitina , Sulfatos de Condroitina , Glioma , Proteínas de Membrana , Humanos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/metabolismo , Glioma/metabolismo , Glioma/patologia , Integrina alfaV , Proteínas de Membrana/metabolismo , Microambiente TumoralRESUMO
It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.
Assuntos
Distrofia Muscular de Duchenne , Humanos , Animais , Camundongos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofina/genética , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Autofagia/genética , Modelos Animais de DoençasRESUMO
Results of experimental studies have shown that ß-aminoisobutyric acid (BAIBA), an exercise-induced myokine-like molecule, is an endogenous negative regulator of fat mass in mice, but it remains unclear whether that is the case in humans, though an enhanced BAIBA concentration in patients receiving sodium-glucose cotransporter 2 inhibitors was found in our recent study. The objective of this study was to analyze the determinants of circulating BAIBA concentration in humans, with focus on the possible link between circulating BAIBA and body composition including fat mass. Data for 188 consecutive patients with heart failure (HF, 64 ± 13 years; 70% male) who received a dual energy X ray absorptiometry (DEXA) scan for assessment of body composition including fat mass index (FMI) and appendicular skeletal muscle mass index (ASMI) were used in this study. Plasma BAIBA concentration in a fasting state after stabilization of HF was determined using ultraperformance liquid chromatography. Plasma BAIBA was detected in 66% of the patients. In simple linear regression analyses of data from patients in whom plasma BAIBA was detected, plasma BAIBA concentration was positively correlated with uric acid and was negatively correlated with body mass index (BMI), estimated glomerular filtration rate (eGFR), FMI, and % body fat. There were no correlations between plasma BAIBA concentration and indexes of muscle mass and bone mass. The results of multiple linear regression analyses showed that FMI and % body fat in addition to BMI, but not ASMI, were independent explanatory factors for plasma BAIBA concentration. In conclusion, plasma BAIBA concentration is inversely correlated with indexes of fat mass, indicating that BAIBA may be a therapeutic target for excessive fat accumulation.
Assuntos
Insuficiência Cardíaca , Miocinas , Humanos , Masculino , Camundongos , Animais , Feminino , Índice de Massa Corporal , Ácidos Aminoisobutíricos/farmacologia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/tratamento farmacológicoRESUMO
Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.
RESUMO
Lectin microarray (LMA) is a high-throughput platform that enables the rapid and sensitive analysis of N- and O-glycans attached to glycoproteins in biological samples, including formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we evaluated the sensitivity of the advanced scanner based on the evanescent-field fluorescence principle, which is equipped with a 1× infinity correction optical system and a high-end complementary metal-oxide semiconductor (CMOS) image sensor in digital binning mode. Using various glycoprotein samples, we estimated that the mGSR1200-CMOS scanner has at least fourfold higher sensitivity for the lower limit of linearity range than that of a previous charge-coupled device scanner (mGSR1200). A subsequent sensitivity test using HEK293T cell lysates demonstrated that cell glycomic profiling could be performed with only three cells, which has the potential for the glycomic profiling of cell subpopulations. Thus, we examined its application in tissue glycome mapping, as indicated in the online LM-GlycomeAtlas database. To achieve fine glycome mapping, we refined the laser microdissection-assisted LMA procedure to analyze FFPE tissue sections. In this protocol, it was sufficient to collect 0.1 mm2 of each of the tissue fragments from 5-µm-thick sections, which differentiated the glycomic profile between the glomerulus and renal tubules of a normal mouse kidney. In conclusion, the improved LMA enables high-resolution spatial analysis, which expands the possibilities of its application classifying cell subpopulations in clinical FFPE tissue specimens. This will be used in the discovery phase for the development of novel glyco-biomarkers and therapeutic targets, and to expand the range of target diseases.
Assuntos
Glicoproteínas , Lectinas , Humanos , Animais , Camundongos , Inclusão em Parafina , Células HEK293 , Formaldeído , Fixação de TecidosRESUMO
Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galß1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis.
Assuntos
Hemorragia , Linfonodos , Sialiltransferases , Animais , Camundongos , Antígenos Virais de Tumores/análise , Antígenos Virais de Tumores/metabolismo , Membrana Celular , Linfonodos/irrigação sanguínea , Camundongos Knockout , Hemorragia/genética , Hemorragia/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismoRESUMO
Aging is associated with impairment of multiple organs, including skeletal muscle and heart. In this study, we investigated whether resveratrol, an activator of an NAD+-dependent protein deacetylase Sirtuin-1 (SIRT1), attenuates age-related sarcopenia and cardiomyocyte hypertrophy in mice. Treatment of mice with resveratrol (0.4 g/kg diet) from 28 weeks of age for 32 weeks prevented aging-associated shortening of rotarod riding time. In the tibialis anterior (TA) muscle, histogram analysis showed that the atrophic muscle was increased in 60-week-old (wo) mice compared with 20-wo mice, which was attenuated by resveratrol. In the heart, resveratrol attenuated an aging-associated increase in the cardiomyocyte diameter. Acetylated proteins were increased and autophagic activity was reduced in the TA muscle of 60-wo mice compared with those of 20-wo mice. Resveratrol treatment reduced levels of acetylated proteins and restored autophagic activity in the TA muscle. Aging-related reduction in myocardial autophagy was also suppressed by resveratrol. Skeletal muscle-specific SIRT1 knockout mice showed increases in acetylated proteins and atrophic muscle fibers and reduced autophagic activity in the TA muscle. These results suggest that activation of SIRT1 by treatment with resveratrol suppresses sarcopenia and cardiomyocyte hypertrophy by restoration of autophagy in mice.
Assuntos
Sarcopenia , Estilbenos , Camundongos , Animais , Resveratrol/farmacologia , Sarcopenia/tratamento farmacológico , Sarcopenia/metabolismo , Sirtuína 1/metabolismo , Músculo Esquelético/metabolismo , Envelhecimento , Miócitos Cardíacos/metabolismo , Hipertrofia , Estilbenos/farmacologia , Estilbenos/uso terapêuticoRESUMO
AIMS/INTRODUCTION: We previously showed that upregulation of myocardial adenosine monophosphate deaminase (AMPD) is associated with pressure overload-induced diastolic dysfunction in type 2 diabetes hearts. Here, we examined involvement of AMPD localized in the endoplasmic reticulum-mitochondria interface in mitochondrial Ca2+ overload and its pathological significance. MATERIALS AND METHODS: We used type 2 diabetes Otsuka Long-Evans Tokushima Fatty rats (OLETF) and non-diabetes Long-Evans Tokushima Otsuka Fatty rats (LETO) as well as AMPD3-overexpressing H9c2 cells and human embryonic kidney 293 cells. RESULTS: OLETF, but not LETO, showed diastolic dysfunction under the condition of phenylephrine-induced pressure overload. The levels of 90-kDa AMPD3 in outer mitochondrial membranes/endoplasmic reticulum and mitochondria-associated endoplasmic reticulum membrane (MAM) fractions were significantly higher in OLETF than in LETO. The area of the MAM quantified by electron microscopic analysis was 57% larger, mitochondrial Ca2+ level under the condition of pressure overload was 47% higher and Ca2+ retention capacity in MAM-containing crude mitochondria isolated before the pressure overloading was 21% lower in OLETF than in LETO (all P-values <0.05). Transfection of FLAG-AMPD3 in cells resulted in significant enlargement of the MAM area, and impairment in pyruvate/malate-driven adenosine triphosphate-stimulated and uncoupler-stimulated mitochondrial respiration compared with those in control cells. CONCLUSIONS: The findings suggest that 90-kDa AMPD3 localized in the endoplasmic reticulum-mitochondria interface promotes formation of the MAM, inducing mitochondrial Ca2+ overload and dysfunction in type 2 diabetes hearts.
Assuntos
Diabetes Mellitus Tipo 2 , Ratos , Animais , Humanos , Diabetes Mellitus Tipo 2/patologia , Ratos Endogâmicos OLETF , Ratos Long-Evans , Mitocôndrias/patologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Monofosfato de Adenosina/metabolismoRESUMO
Systemic branched-chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long-Evans-Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched-chain α-keto acid dehydrogenase (BCKDH), a rate-limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long-Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH-E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3-E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3-BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3-BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.
Assuntos
3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida) , AMP Desaminase , Diabetes Mellitus Tipo 2 , Animais , Ratos , AMP Desaminase/genética , AMP Desaminase/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Proteômica , Ratos Endogâmicos OLETF , Ratos Long-Evans , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genéticaRESUMO
Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of cells. METHODS: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. RESULTS: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. CONCLUSIONS: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.
RESUMO
Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism.